Circadian rhythms are a fundamental property of all eukaryotic and some prokaryotic organisms (Takahashi 1995). The underlying molecular mechanism appears similar among living systems, is cell autonomous and involves periodic macromolecular synthesis. Alterations in circadian rhythms are involved in sleep disorders such as "delayed sleep phase syndrome" which may be an alteration in the circadian period (lengthening) and the entrainment system. There is also evidence for circadian rhythm abnormalities in affective disorders. The most consistent feature of circadian rhythms observed in depressed patients is that a variety of physiological events occur earlier than normal (usually referred to as a "phase advance"). A shortened REM latency after sleep onset, which can be the manifestation of a change in the circadian coupling or organization of rhythms, appears to be a prominent characteristic of depression.
Further, a number of diagnostic tests depend on the time of day at which the test is performed. These include the dexamethasone suppression test for depression, intraocular pressure measurements for glaucoma, and plasma cortisol concentration for Addison's disease and Cushing's syndrome. In addition, a number of clinical treatments (such as chemotherapy or alleviation of hypertension) can be optimized through the delivery of therapeutic agents at the appropriate time of day. Circadian rhythmicity appears to be deeply embedded in most aspects of the biology of organisms--indeed it is a central feature of their organization. It seems unlikely that complete understanding of most regulatory processes can be achieved without an appreciation of their circadian dimensions.
Clock genes have been described in other model systems, most notably in Drosophila and Neurospora. Three known clock genes have been characterized at the molecular and functional level. These are the period (per) and timeless (tim) genes in Drosophila, and the frequency (frq) gene in Neurospora. This work is known to the art and is described in review papers by J. S. Takahashi, Annual Review of Neuroscience 18:531-553, 1995; and by J. C. Dunlap, Annual Review of Genetics 30:579-601, 1996. None of these three clock genes have been shown to possess a protein motif known to allow these proteins to bind DNA, rather it appears that in the case of PERIOD and TIMELESS, these proteins must interact with unidentified DNA-binding transcription factors.
The genetic approach to circadian rhythms was first described by Ron Konopka and Seymour Benzer (1971) who isolated single-gene mutations that altered circadian periodicity in Drosophila. In a chemical mutagenesis screen of the X chromosome, they found three mutants that either shortened (per.sup.S), lengthened (per.sup.L) or abolished (per.sup.0) circadian rhythms of eclosion and adult locomotor activity. In 1984, two groups at Brandeis and Rockefeller independently cloned per in a series of experiments that showed that germline transformation with DNA could rescue a complex behavioral program (reviewed in Rosbash & Hall 1989). Each of the mutant per alleles is caused by intragenic point mutations that produce missense mutations in per.sup.S and per.sup.L and a nonsense mutation in per.sup.0 (Bayfies et al. 1987, Yu et al. 1987). Only recently has the nature of per gene product (PER )become more clear. The Drosophila single-minded protein (SIM) (Nambu et al. 1991), the human aryl hydrocarbon receptor nuclear translocator (ARNT) (Hoffirian et al. 1991), and the aryl hydrocarbon receptor (AHR) (Burbach et al. 1992) all share with PER a domain called PAS (for PER, ARNT, SIM) (Nambu et al. 1991). The PAS domain contains about 270 amino acids of sequence similarity with two 51-amino acid direct repeats. Recent work shows that the PAS domain can function as a dimerization domain (Huang et al. 1993). Because other PAS members are transcriptional regulators and PER can dimerize to them, PER could function as a transcriptional regulator either by working in concert with apartner that carries a DNA-binding domain, or by acting as a dominant-negative regulator by competing with a transcriptional regulator for dimenization or DNA binding. Consistent with this role, PER is predominantly a nuclear protein in the adult central nervous system of Drosophila (Liu et al. 1992).
The expression of PER itself is circadian, and both per mRNA and PER protein abundance levels oscillate. Hardin et al. (1990) showed that per mRNA levels undergo a striking circadian oscillation. The per RNA rhythm persists in constant darkness and the period of the RNA rhythm is .about.24 hours in per.sup.+ flies and is .about.20 hours in per.sup.S flies. The RNA of per.sup.0 flies is present at a level .about.50% of normal flies, but does not oscillate. In per.sup.0 flies that have been rescued by gernnline transformation with wild-type per.sup.+ DNA, both circadian behavior and per RNA cycles are restored. Importantly, in these transformed flies both the exogenous per.sup.+ RNA and the endogenous per.sup.0 RNA levels oscillate. In addition to a per RNA cycle, the PER protein also shows a circadian rhythm in abundance (Siwicki et al. 1988, Zerr et al. 1990, Edery et al. 1994b). The rhythm in PER protein also depends on per, because per.sup.0 flies do not have a protein rhythm and because per mutants alter the PER rhythm (Zerr et al. 1990). Therefore, the circadian expression of per mRNA and protein levels both depend on an active per gene. Because per.sup.S shortens the period of the RNA cycle and because per.sup.+ DNA transformation rescues per.sup.0 RNA cycling, PER protein expression clearly regulates per RNA cycling. Hardin et al. (1990) propose that feedback of the per gene product regulates its own mRNA levels. Support for such a model has been provided by showing that transient induction of PER from a heat shock promoter/per cDNA transgene in a wild-type background can phase shift circadian activity rhythms in Drosophila (Edery et al. 1994a).
The PER protein rhythm appears to be regulated at both transcriptional and post-transcriptional levels. Hardin et al. (1992) have shown that levels of per precursor RNA cycle in concert with mature per transcripts. In addition, per promoter/CAT fusion gene constructs show that per 5' flanking sequences are sufficient to drive heterologous RNA cycles. These results suggest that circadian fluctuations in per mRNA abundance are controlled at the transcriptional level. In addition to a rhythm in per transcription and PERabundance, PER appears to undergo multiple phosphorylation events as itaccumulates each cycle (Edery et al. 1994b). The nature and functional significance of the PER phosphorylation sites, however, are not known at this time. Interestingly, the peak of the per RNA cycle precedes the peak of the PER protein cycle by about 4-6 hours. The reasons for the lag in PER accumulation are not well understood. However, the recent isolation of a second clock mutant, named timeless (tim), has provided significant insight (Sehgal et al. 1994). Tim mutants fail to express circadian rhythms in eclosion and locomotor activity, but more importantly also fail to express circadian rhythms in per mRNA abundance (Sehgal et al. 1994). Furthermore, the nuclear localization of PER is blocked in tim mutants (Vosshall et al. 1994). In 1995, tim was cloned by positional cloning and by interaction with the PAS domain of PER in a yeast two-hybrid screen (Gekakis et al. 1995, Myers et al. 1995). Like PER, TIM is a large protein without any obvious sequence homologies to other proteins. While PER dimerizes to TIM via the PAS domain, TIM is not a member of the PAS family. The expression of tim RNA levels has a striking circadian oscillation which is in phase with the per RNA rhythm. The rhythm in tim RNA levels depends on PER and is abolished in per.sup.0 mutants and shortened in per.sup.S mutants. Thus, per and tim express a coordinate circadian rhythm that is interdependent. TIM protein also shows a circadian rhythm with a phase similar to that of PER. Formation of a PER/TIM heterodimer appears to be required for nuclear entry of the complex. In the last year, four different laboratories discovered that light exposure causes a rapid degradation of TIM protein in flies and this action of light can explain how entrainment of the circadian clock in Drosophila occurs (Hunter-Ensor et al. 1996, Lee et al. 1996, Myers et al. 1996, Zeng et al. 1996). Thus, the identification of tim and its functional interaction with per is important because it suggests that elements of a transcription-translation-nuclear transport feedback loop are central elements of the circadian mechanism in Drosophila.
In addition to the Drosophila per and tim genes, progress has been made in elucidating the molecular nature of the Neurosporafrequency (frq) gene (Dunlap 1993). Like per, the frq locus is defined by mutant alleles that either shorten, lengthen or disrupt circadian rhythms (Feldman & Hoyle 1973, Feldman 1982, Dunlap 1993). Cloned in 1989, the sequence of FRQ shows little resemblance to PER (except for a region containing threonine-glycine repeats) (McClung et al. 1989); however, recent molecular work shows striking functional similarities (Aronson et al. 1994). The frq gene expresses a circadian oscillation of mRNA abundance whose period is altered by frq mutations (Aronson et al. 1994). A null allele, frq.sup.9, expresses elevated levels of frq transcript and does not show a rhythm in mRNA abundance (Aronson et al. 1994). Interestingly, no level of constitutive expression of frq.sup.+ in a null background can rescue overt rhthmicity, which suggests that the circadian rhythm of frq mRNA is a necessary component of the oscillator (Aronson et al. 1994). However, overexpression of a frq.sup.+ transgene does negatively autoregulate expression of the endogenous of a frq gene (Aronson et al. 1994). In addition, overexpression of frg.sup.+ transgene in a wild-type background blocks overt expression of circadian rhythms (Aronson et al. 1994). The phase of the overt circadian rhythm can be determined by a step reduction in FRQ protein expression (Aronson et al. 1994). Taken together, these experiments show that frq is likely a central component of the Neurospora circadian oscillator and that a negative autoregulatory loop regulating frq transcription forms the basis of the oscillation (Aronson et al. 1994). Recently a direct action of light has been found on frq expression (Crosthwaite et al. 1995). Frq transcription is rapidly induced by light exposure and this effect of light can explain photic entrainment in Neurospora in a simple and direct manner.
Although there are remarkable functional similarities between per and frq, there are also distinct differences. The phases of the mRNA rhythms are different: per peaks at night (Hardin et al. 1990), whereas frq peaks during the day (Aronson et al. 1994). While per overexpression shortens circadian period (Smith & Konopka 1982, Baylies et al. 1987), frq overexpression does not change period but rather abolishes overt rhythmicity (Aronson et al. 1994). The null allele, per.sup.0, leads to a constant level of mRNA that is about 50% of the peak level of wild-type levels in Drosophila (Hardin et al. 1990); while in Neurospora, frq.sup.9 mRNA levels are significantly elevated relative to wild-type (Aronson et al. 1994). Finally, the action of light on these two systems is opposite: light degrades TIM protein in Drosophila; whereas, it activates the transcription of frq in Neurospora. These differences can be interpreted in at least two ways: 1) the elements of each system are not fully defined and frq and per could define different elements in a conserved pathway within the oscillator feedback loop; or 2) the Drosophila and Neurospora circadian clocks could be functionally analogous rather than phylogenetically homologous. Irrespective of the interpretation, however, it appears likely that a transcription-translation autoregulatory feedback loop may be a common feature of circadian clocks.
Searching for per homologs in mammals has not been very productive despite ten years of effort by a number of laboratories. This is probably due to the relatively low level of sequence similarity of per even among the Drosophilids (Hall 1990). Putative per homologs in mammals have been reported in searches directed against the threonine-glycine (TG) repeat region of PER (Shin et al. 1985, Matsui et al. 1993) and the region of the per.sup.S mutation (Siwicki et al. 1992). However, the TG-repeat clones show no other sequence similarity to PER, and the antigenes detected by antibodies to the per.sup.S region have not been characterized molecularly. New efforts targeted against the PAS dimerization domain (Huang et al. 1993), which is moderately well-conserved among insects (Reppert et al. 1994), using either PCR-based approaches or the yeast two-hybrid system (Fields & Song 1989) could eventually succeed as more bona fide per homologs are cloned in species more closely related to insects. Alternatively, as other Drosophila clock genes are cloned in the future, some should have sequence conservation with mammals as found, for example, with genes regulating pattern formation (Krumlauf 1993) or signal transduction (Zipursky & Rubin 1994). However, at this time no confirmed orthologs of per, tim or frq have been cloned in any vertebrate.
Very little information on the genetics of mammalian circadian rhythms is available. Most work in the field has used quantitative genetic approaches such as comparisons of circadian phenotype among inbred strains of mice and rats, recombinant inbred strain analysis, or selection of natural variants (Hall 1990, Schwartz & Zimmerman 1990, Lynch & Lynch 1992). The most comprehensive analysis of inbred mouse strains was done by Schwartz & Zimmerman (1990) who compared 12 different strains and found that the most extreme strains (C57BL/6J and BALB/cByJ) had a period difference of about one hour in constant darkness. Reciprocal F1 hybrid and recombinant inbred strain analysis provided no evidence of monogenic inheritance of the circadian period. Polygenic inheritance of circadian traits (or more strictly, failure to detect monogenic inheritance) has been the conclusion of every quantitative genetic analysis performed thus far.
A notable exception to the general finding of polygenic control of circadian phenotype is the spontaneous mutation, tau, found in the golden hamster (Ralph & Menaker 1988). Tau is a semidommant, autosomal mutation that shortens circadian period by two hours in heterozygotes and by four hours in homozygotes. Its phenotype is remarkably similar to the Drosophila per.sup.S allele being semidominant, changing period to the same extent, and increasing the amplitude of the phase response curve to light (Ralph & Menaker 1988, Ralph 1991). The tau mutation has been extremely useful for physiological analysis. For example, the circadian pacemaker function of the suprachiasmatic nuclei (SCN) has been definitively demonstrated by transplantation of SCN tissue derived from tau mutant hamsters to establish that the genotype of the donor SCN determines the period of the restored rhythm (Ralph et al. 1990). Furthermore, the effects of having both tau mutant and wild-type SCN tissue in the same animal show that both mutant (.about.20 h) and wild-type (.about.24 h) periodicities can be expressed simultaneously suggesting that very little interaction of the oscillators occurs under these conditions (Vogelbaum & Menaker 1992). Additional cellular interactions can also be studied by transplantation of dissociated SCN cells derived from tau mutant and wild-type animals (Ralph & Lehman 1991). Thus, a number of issues that could not be addressed previously have been resolved or approached by the use of the tau mutation.
Unfortunately, not much progress has been made on the genetic and molecular nature of tau. Genetic mapping and molecular cloning of tau remains difficult because of the paucity of genetic information in the golden hamster. Thus far the tau mutation has contributed substantially to physiological analysis, but it will be difficult to elucidate the nature of the tau gene product unless candidate genes become apparent or the hamster is developed as a genetic system.